Download E.Z.N.A.®SP Plant DNA Maxi Kit - Omega Bio-Tek

E.Z.N.A.® SP Plant DNA Maxi Kit
D5538-00
D5538-01
2 preps
5 preps
February 2014
E.Z.N.A.® SP Plant DNA Maxi Kit
Table of Contents
Introduction and Overview.......................................................2
Kit Contents/Storage and Stability.........................................3
Preparing Reagents.......................................................................4
Sample Preparation......................................................................5
SP Plant DNA Maxi Protocol - Dried Samples......................6
SP Plant DNA Maxi Protocol - Fresh/Frozen Samples.....10
Troubleshooting Guide.............................................................14
Ordering....................................................................................15
Manual Revision: February 2014
Innovations in nucleic acid isolation
1
Introduction and Overview
The E.Z.N.A.® SP Plant DNA Maxi Kit is specially designed for rapid and reliable isolation
of high-quality total cellular DNA from plant species containing high levels of phenolic
compounds and polysaccharides. Up to 1 g wet tissue (or 250 mg dry tissue) can be
processed in less than 1 hour. The system combines the reversible nucleic acid-binding
properties of the HiBind® matrix with the speed and versatility of spin column technology
to eliminate polysaccharides, phenolic compounds, and enzyme inhibitors from plant
tissue lysates. Purified DNA is suitable for PCR, restriction digestion, and hybridization
techniques.
If using the E.Z.N.A.® SP Plant DNA Maxi Kit for the first time, please read this booklet
to become familiar with the procedures. Dry or fresh/frozen plant tissue is disrupted
and lysed in a specially formulated buffer containing detergent. Binding conditions are
adjusted and the sample is transferred to a HiBind® DNA Maxi Column. Two rapid wash
steps remove trace contaminants such as residual polysaccharides, and pure DNA is eluted
in water or low ionic strength buffer. Purified DNA can be directly used in downstream
applications without the need for further purification.
New in this Edition:
•
Equilibration Buffer is no longer included with this kit. An optional Column
Equilibration Protocol has been added to the protocol for your convenience.
•
Equilibration Buffer is replaced with 3M NaOH provided by the user.
Binding Capacity: Each HiBind® DNA Maxi Column can bind up to 2 mg genomic DNA.
Using more than 2 g fresh plant tissue is not recommend.
2
Kit Contents
Product
D5538-00
D5538-01
Purifications
2
5
HiBind® DNA Maxi Columns
2
5
Homogenizer Maxi Columns
2
5
50 mL Collection Tubes*
4
10
SP1 Buffer
20 mL
40 mL
SP2 Buffer
6 mL
20 mL
SP3 Buffer
10 mL
40 mL
SPW Wash Buffer
5 mL
25 mL
Elution Buffer
5 mL
80 mL
User Manual
P
P
* HiBind® DNA Maxi Columns and Homogenizer Columns have been inserted into the 50
mL Collection Tubes for your convenience.
Storage and Stability
All of the E.Z.N.A.® SP Plant DNA Maxi Kit components are guaranteed for at least 12
months from the date of purchase when stored as follows. All components should be
stored at room temperature. During shipment or storage in cool ambient conditions,
precipitates may form in SP1 Buffer and/or SP3 Buffer. Dissolve such deposits by warming
the solution at 37°C and gently shaking.
3
Preparing Reagents
1.
2.
Dilute SP3 Buffer with 100% ethanol as follows and store at room temperature.
Kit
100% Ethanol to be Added
D5538-00
20 mL
D5538-01
80 mL
Dilute SPW Wash Buffer with 100% ethanol as follows and store at room temperature.
Kit
4
100% Ethanol to be Added
D5538-00
20 mL
D5538-01
100 mL
Sample Preparation
Choose the most appropriate protocol. Procedures are described for both dried and fresh/
frozen samples.
Sample Type
Page Number Starting Amount
Dried
5
250 mg powdered tissue
Fresh/Frozen
10
1 g tissue
Recommended Sample Preparation
1.
Dried Specimens
Drying allows storage of field specimens for prolonged periods of time prior to
processing. Samples can be dried overnight in a 45°C oven, powdered, and stored
dry at room temperature. To prepare dried samples, place up to 250 mg dried tissue
into a 50 mL centrifuge tube and grind using a pellet pestle. For critical work such as
PCR and cloning, pestles are best used a single time then soaked in a dilute bleach
solution immediately after use until clean. For standard Southern analysis, the same
pestle can be reused several times to grind multiple tissue samples by rinsing with
ethanol and carefully wiping the surfaces clean between samples. Disposable pestles
may be autoclaved several times. A fine powder will ensure optimal DNA extraction
and yield.
2.
Fresh/Frozen Specimens
Due to the tremendous variation in water and polysaccharide content of plants,
sample size should be limited to 1 g for first time users. It is very important to not
overload the HiBind® DNA Maxi Column. Too much starting material will decrease the
yield and purity due to inefficient lysis. However, for some plant species, increasing
the starting material can increase DNA yield. We recommend starting with 1 g tissue.
If results obtained are satisfactory, then increase amount of starting material. Best
results are obtained with young leaves or needles.
Although various means of sample disruption can be used for this kit, such as
beads or pestles, we recommend grinding the sample in liquid nitrogen. To prepare
samples, collect tissue in a 30 mL mortar and freeze by adding liquid nitrogen. Grind
the tissue using a clean pestle. Alternatively, allow the liquid nitrogen to evaporate
and store the samples at -70°C for later use. For critical work such as PCR and
cloning, pestles are best used a single time then soaked in a dilute bleach solution
immediately after use until clean. For standard Southern analysis, the same pestle can
be reused several times to grind multiple tissue samples by rinsing with ethanol and
carefully wiping the surfaces clean between samples. Transfer the ground sample
into a 50 mL centrifuge tube.
Note: Do not allow the sample to thaw during handling and weighing. To prevent
the sample from thawing, keep the tubes on a bed of dry ice.
5
E.Z.N.A.® SP Plant DNA Maxi Kit Protocols
E.Z.N.A.® SP Plant DNA Maxi Kit Protocol - Dried Samples
Materials and Equipment to be Supplied by User:
•
•
•
•
•
•
•
•
Centrifuge capable of at least 3,000 x g
Vortexer
Nuclease-free 50 mL centrifuge tubes capable of withstanding 3,000 x g
Incubator, heat block, or water bath capable of 65°C
100% ethanol
RNase A solution at 50 mg/mL
Ice bucket
Optional for fresh samples: liquid nitrogen for freezing/grinding samples
Before Starting:
•
•
•
•
•
Prepare SPW Wash Buffer and SP3 Buffer according to the Preparing Reagents section
on Page 4
Set an incubator, heat block, or water bath to 65°C
Heat Elution Buffer to 65°C
Prepare an ice bucket
Prepare an RNase A solution at 50 mg/mL
1.
Transfer up to 250 mg dried powdered tissue to a 50 mL centrifuge tube.
2.
Add 7 mL SP1 Buffer and 20 μL RNase A solution (50 mg/mL). Vortex at maximum
speed to mix thoroughly. Do not mix SP1 Buffer and RNase A before use.
Note: Ensure that all the samples are completely suspended and that there are no
clumps in the solution. Clumps will result in low yields.
3.
Incubate at 65°C for 30-60 minutes. Invert the tube several times during incubation
to mix.
4.
Add 2.5 mL SP2 Buffer. Vortex to mix thoroughly.
5.
Let sit on ice for 10 minutes.
6
E.Z.N.A.® SP Plant DNA Maxi Kit Protocols
6.
Centrifuge at maximum speed (≥ 3,000 x g) for 6 minutes at room temperature.
Note: Some plant materials can generate very viscous lysates and large amounts
of precipitates during this step. The preparation of a cleared lysate is essential to
prevent clogging of the HiBind® DNA Maxi Column. For some plant samples, it is
difficult to clear the lysate via a single centrifugation step because not all particulate
matter forms a compact pellet. Omega Homogenizer Maxi Columns can efficiently
remove most cell debris and precipitates in next step.
7.
Carefully transfer the supernatant to a Homogenizer Maxi Column. Do not disturb the
pellet.
8.
Centrifuge at maximum speed for 5 minutes.
Note: Longer centrifugation does not improve yield. The Homogenizer Column will
remove most precipitates and cell debris, but a small amount might pass through
and form a pellet in the Collection Tube.
9.
Carefully transfer the cleared lysate to a new 50 mL centrifuge tube. Do not disturb
the pellet. Measure the volume of the lysate for the next step.
10. Add 1.5 volumes SP3 Buffer. Immediately vortex to obtain a homogeneous mixture. If
precipitation can be seen at this point, break the precipitation by passing through a
needle using a syringe or pipetting up and down 10-15 times.
Important: Reserve the lysate for later use. Do not use in Step 11.
Note: SP3 Buffer must be diluted with ethanol before use. Please see the Preparing
Reagents section on Page 4 for instructions.
Optional Protocol for Column Equilibration:
Important: Do not add 3M NaOH to the lysate from Step 10.
1.
2.
3.
4.
Add 3 mL 3M NaOH to the HiBind® DNA Maxi Column.
Let sit at room temperature for 4 minutes.
Centrifuge at 3,000 x g for 3 minutes.
Discard the filtrate and reuse the collection tube.
7
E.Z.N.A.® SP Plant DNA Maxi Kit Protocols
11. Transfer 15 mL lysate (including any precipitation) from Step 10 to the HiBind® DNA
Maxi Column.
12. Centrifuge at maximum speed for 5 minutes.
13. Discard the filtrate and reuse the Collection Tube.
14. Repeat Steps 11-13 until all of the lysate has been transferred to the column.
15. Add 10 mL SPW Wash Buffer.
Note: SPW Wash Buffer must be diluted with ethanol before use. Please see the
Preparing Reagents section on Page 4 for instructions.
16. Centrifuge at maximum speed for 4 minutes.
17. Discard the filtrate and reuse the Collection Tube.
18. Repeat Steps 15-17 for a second SPW Wash Buffer wash step.
19. Centrifuge the empty column at maximum speed for 10 minutes.
Note: It is important to dry the column membrane before elution. Residual ethanol
may interfere with downstream applications.
20. Transfer the HiBind® DNA Maxi Column to a 50 mL centrifuge tube (not provided).
21. Add 2 mL Elution Buffer heated to 65°C directly to the center of column matrix.
22. Let sit at room temperature for 5 minutes.
23. Centrifuge at maximum speed for 3 minutes.
8
E.Z.N.A.® SP Plant DNA Maxi Kit Protocols
24. Repeat Steps 21-23 for a second elution step.
Note: This step may be performed using another 50 mL centrifuge tube to maintain a
higher DNA concentration in the first eluate. Alternatively, DNA concentration can be
increased by using the first eluate for the second elution step.
25. Store DNA at -20°C.
9
E.Z.N.A.® SP Plant DNA Maxi Kit Protocols
E.Z.N.A.® SP Plant DNA Maxi Kit Protocol - Fresh/Frozen
Specimens
Materials and Equipment to be Supplied by User:
•
•
•
•
•
•
•
•
Centrifuge capable of at least 3,000 x g
Vortexer
Nuclease-free 50 mL centrifuge tubes capable of withstanding 3,000 x g
Incubator, heat block, or water bath capable of 65°C
100% ethanol
RNase A solution at 50 mg/mL
Ice bucket
Optional for fresh samples: liquid nitrogen and mortar and pestle for freezing and
grinding samples
Before Starting:
•
•
•
•
•
Prepare SPW Wash Buffer and SP3 Buffer according to the Preparing Reagents section
on Page 4
Set an incubator, heat block, or water bath to 65°C
Heat Elution Buffer to 65°C
Prepare an ice bucket
Prepare an RNase A solution at 50 mg/mL
1.
Transfer up to 1 g ground plant tissue to a 50 mL centrifuge tube (not provided).
2.
Add 5 mL SP1 Buffer and 20 μL RNase A stock solution (50 mg/mL). Vortex at
maximum speed to mix thoroughly. Do not mix SP1 Buffer and RNase A before use.
Note: Ensure that all the samples are completely suspended and that there are no
clumps in the solution. Clumps will result in low yields.
3.
Incubate at 65°C for 30 minutes. Invert the tube several times during incubation to
mix.
4.
Add 1.75 mL SP2 Buffer. Vortex to mix thoroughly.
5.
Let sit on ice for 10 minutes.
10
E.Z.N.A.® SP Plant DNA Maxi Kit Protocols
6.
Centrifuge at maximum speed (≥ 3,000 x g) for 6 minutes at room temperature.
Note: Some plant materials can generate very viscous lysates and large amounts
of precipitates during this step. The preparation of a cleared lysate is essential to
prevent clogging of the HiBind® DNA Maxi Column. For some plant samples, it is
difficult to clear the lysate via a single centrifugation step because not all particulate
matter forms a compact pellet. Omega Homogenizer Maxi Columns can efficiently
remove most cell debris and precipitates in next step.
7.
Carefully transfer the supernatant to a Homogenizer Maxi Column. Do not disturb the
pellet.
8.
Centrifuge at maximum speed for 5 minutes.
Note: Longer centrifugation does not improve yield. The Homogenizer Column will
remove most precipitates and cell debris, but a small amount might pass through
and form a pellet in the Collection Tube.
9.
Carefully transfer the cleared lysate to a new 50 mL centrifuge tube. Do not disturb
the pellet. Measure the volume of the lysate for the next step.
10. Add 1.5 volumes SP3 Buffer. Immediately vortex to obtain a homogeneous mixture. If
precipitation can be seen at this point, break the precipitation by passing through a
needle using a syringe or pipetting up and down 10-15 times.
Important: Reserve the lysate for later use. Do not use in Step 11.
Note: SP3 Buffer must be diluted with ethanol before use. Please see the Preparing
Reagents section on Page 4 for instructions.
Optional Protocol for Column Equilibration:
Important: Do not add 3M NaOH to the lysate from Step 10.
1.
2.
3.
4.
Add 3 mL 3M NaOH to the HiBind® DNA Maxi Column.
Let sit at room temperature for 4 minutes.
Centrifuge at 3,000 x g for 3 minutes.
Discard the filtrate and reuse the collection tube.
11
E.Z.N.A.® SP Plant DNA Maxi Kit Protocols
11. Transfer 15 mL lysate (including any precipitation) from Step 10 to the HiBind® DNA
Maxi Column.
12. Centrifuge at maximum speed for 5 minutes.
13. Discard the filtrate and reuse the Collection Tube.
14. Repeat Steps 11-13 until all of the lysate has been transferred to the column.
15. Add 10 mL SPW Wash Buffer.
Note: SPW Wash Buffer must be diluted with ethanol before use. Please see the
Preparing Reagents section on Page 4 for instructions.
16. Centrifuge at maximum speed for 4 minutes.
17. Discard the filtrate and reuse the Collection Tube.
18. Repeat Steps 15-17 for a second SPW Wash Buffer wash step.
19. Centrifuge the empty column at maximum speed for 10 minutes.
Note: It is important to dry the column membrane before elution. Residual ethanol
may interfere with downstream applications.
20. Transfer the HiBind® DNA Maxi Column to a 50 mL centrifuge tube (not provided).
21. Add 2 mL Elution Buffer heated to 65°C directly to the center of column matrix.
22. Let sit at room temperature for 5 minutes.
23. Centrifuge at maximum speed for 3 minutes.
12
E.Z.N.A.® SP Plant DNA Maxi Kit Protocols
24. Repeat Steps 21-23 for a second elution step.
Note: This step may be performed using another 50 mL centrifuge tube to maintain a
higher DNA concentration in the first eluate. Alternatively, DNA concentration can be
increased by using the first eluate for the second elution step.
25. Store DNA at -20°C.
13
Troubleshooting Guide
Please use this guide to troubleshoot any problems that may arise. For further assistance,
please contact the technical support staff, toll free, at 1-800-832-8896.
Problem
Clogged
column
Cause
Solution
Debris carryover
Following precipitation with SP2 Buffer, make
sure no particulate material is transferred.
DNA pellet not
completely dissolved
before applying
sample to column
Ensure that DNA is dissolved in water before
adding SP3 Buffer. This may need repeated
incubation at 65°C and vortexing.
Sample too viscous
Do not exceed suggested amount of starting
material. Alternatively, increase amounts of
SP1 Buffer and SP2 Buffer.
Incomplete
Increase RCF or centrifugation time after
precipitation following
addition of SP2 Buffer.
addition of SP2 Buffer
Problem
Low DNA
yield
Problem
Problems in
downstream
applications
14
Cause
Solution
Incomplete disruption
of starting material
For both dried and fresh/frozen samples,
obtain a fine homogeneous powder before
adding SP1 Buffer.
Poor lysis of tissue
Decrease amount of starting material or
increase amount of SP1 Buffer and SP2 Buffer.
DNA remains bound to Increase elution volume and incubate column
column
at 65°C for 5 minutes before centrifugation.
DNA washed off
Dilute SPW Wash Buffer by adding the
appropriate volume of 100% ethanol prior to
use (Page 4).
Column matrix lost
binding capacity
during storage
Follow the Optional Protocol for Column
Equilibration prior to transferring the cleared
lysate to the HiBind® DNA Mini Column. Add
3 mL 3M NaOH to the column prior to loading
the sample. Let sit for 4 minutes. Centrifuge at
3,000 x g for 3 minutes. Discard the filtrate.
Cause
Solution
Salt carryover
SPW Wash Buffer must be at room
temperature.
Ethanol carryover
Following the second wash step, ensure
that the column is dried by centrifugation at
maximum speed for 10 minutes.
Ordering Information
The following components are available for purchase separately.
(Call Toll Free at 1-800-832-8896)
Product
Part Number
SP1 Buffer, 250 mL
PD086
SP2 Buffer, 60 mL
PD073
SP3 Buffer, 100 mL
PD074
SPW Wash Buffer, 25 mL
PDR045
Elution Buffer, 100 mL
PDR048
HiBind®, E.Z.N.A.®, and MicroElute® are registered trademarks of Omega Bio-tek, Inc.
PCR is a patented process of Hoffman-La Roche. Use of the PCR process requires a license.
15
Notes:
16